Gene expression analysis
Relative gene expression analysis was performed through total RNA extraction and quantitative real-time PCR (qRT-PCR) analysis following our previous operations.30 Briefly, fresh leaves were ground into powder in liquid nitrogen and total RNA was extracted using RNA purification kit (Invitrogen Inc., CA, USA). Following concentration measurement by ultraviolet spectrophotometer (Cary 50, Varian, USA) and integrity determination by agarose gel electrophoresis, a reverse transcription procedure was performed with the M-MLV reverse transcriptase (TaKaRa, Dalian, China) and the resulted cDNA mixture was used for subsequent PCRs analysis.
The qRT-PCR analysis was carried out in a Rotor-gene-6000 real-time PCR system (Corbett Research, Mortlake, Australia). The selected genes and designed primers were listed in Supplementary Table S2. After the preparation of the reaction mixture containing primers, cDNA and SYBR Green Master (ROX, Mannheim, Germany), the PCR was performed under the following temperature procedure: 10 min at 94°C, followed by 40 cycles of 30 s at 94°C, 30 s at 52–55°C (based on the Tm of the primers) and 20 s at 72°C. The relative abundance of gene expression were determined by comparative threshold cycle (Ct) method and actin was used as an internal control.31Three replicates were performed for each sample.